Y-carboxyglutamate derivative, method for preparing the same and method for preparing human osteocalcin using the same

ABSTRACT

Gla 17  human osteocalcin and Glu 17  human osteocalcin or salts thereof are prepared by introducing γ-carboxyglutamic acid into the amino acid sequence of said osteocalcin by employing a protective L-γ-carboxyglutamic acid represented by the formula: ##STR1## wherein n represents 0, 1 or 2, or a salt thereof in the appropriate position in the reacting sequence of amino acids which form the osteocalcin.

BACKGROUND OF THE INVENTION

This invention relates to a novel amino acid derivative, a method forpreparing the same and a method for preparing human osteocalcin usingthe same which is an important index of bone metabolism and aging ofhuman being.

Heretofore, as γ-carboxyglutamic acid derivatives as a starting materialfor chemical synthesis of partial fragment of a protein and a peptide,the following has been known. ##STR2## wherein X, R and R' eachrepresent a combination as shown in the following table.

    ______________________________________                                        Compound   X             R      R'                                            ______________________________________                                        a          Cbz           CH.sub.3                                                                             tBu                                           b          Cbz           Bzl    tBu                                           c          Cbz           CH.sub.3                                                                             Bzl                                           d          tBoc          CH.sub.3                                                                             Bzl                                           e          Cbz           Phac   tBu                                           ______________________________________                                    

wherein Cbz represents a benzyloxycarbonyl group, tBu represents at-butyl group, Bzl represents a benzyl group, tBoc represents at-butyloxycarbonyl group and Phac represents a phenacyl group.

However, when the above compound is used as a starting material for thecompounds shown in the above table, using the benzyloxycarbonyl (Cbzl)group as the α-amino protective group X is possible only in the case ofthe liquid phase method and it is not used or cannot be used (Compoundsa, b, c and e) in the solid phase method or partial solid phase method.Also, regarding the compound d, when it is used in the solid phasemethod and in the partial solid phase method, a problem which exist isthat selectively eliminating of only the α-carboxyl protective group Rwhile leaving the protective groups R intact and R' is impossible.

The present inventors have found that in the above compound representedby the formula, a compound represented by the formula (I) mentionedbelow can be selectively deprotected for the above groups X, R and R'can be utilized in the solid phase method can be the partial solid phasemethod.

Osteocalcin (Bone Gla Protein: BGP) is a vitamin K dependent calciumbindable protein comprising 15 to 20% of the non-collagenic proteins ofbone, and has been considered to be intimately related to both boneformation and bone absorption [Journal of Bone Metabolism Society ofJapan 4, 56, 1986]. Poser et al analyzed the primary structure of humanosteocalcin, and reported that human osteocalcin is a mixture of Glu⁷osteocalcin with the 17-position being glutamic acid and Gla⁷osteocalcin with the 17-position being γ-carboxyglutamic acid having theamino acid sequence shown below existing at a ratio of 91:9 [Poser, J.W. et al., Proc. Natl. Acad. Sci. U.S., 255, 8685-8691 (1980)]. ##STR3##wherein X represents γ-carboxyglutamic acid residue (Gla) or glutamicacid residue (Glu).

However, osteocalcins of cattle, swordfish, cat, chicken, rat, goat, pigand rat already known up to date are all γ-carboxyglutamic acid at thecorresponding site. As the reason, Poser et al estimate that calcifiedbone of aged people is used as the extraction material of humanosteocalcin, and glutamic acid at the 17-position will change toγ-carboxyglutamic acid as man is older.

From such standpoint, establishment of human osteocalcin measuringsystem is not only clinically useful for diagnosis of bone diseases suchas Paget's disease, bone metastasis, etc., but also can be expected toexamine the relationship with aging by preparing the antibodies andantisera which recognize individually the 17-position Gla and Glu ofhuman osteocalcin.

However, there exist none of measuring systems for quantitatingseparately Gla⁷ human osteocalcin and Glu⁷ human osteocalcin as a matterof course, and also human osteocalcin measuring system by use of humanosteocalcin as the standard product. This is because Gla⁷ humanosteocalcin and Glu⁷ human osteocalcin for specific antibody orantiserum preparation, and also as the standard product are notavailable.

In the present specification, the abbreviations, abbreviated symbolsemployed have the following meanings.

1 AMINO ACIDS

Ala: alanine, Arg: arginine, Asn: asparagine, Asp: aspartic acid,Cys:cysteine, Gla: γ-carboxyglutamic acid, Gln: glutamine, Glu: glutamicacid, Gly: glycine, His: histidine, Ile: isoleucine, Leu: leucine, Phe:phenylalanine, Pro: proline, Trp: tryptophan, Tyr: tyrosine and Val:valine.

In some cases, the respective abbreviations may show corresponding aminoacid residues.

2. PROTECTIVE GROUPS

Boc: t-butyloxycarbonyl, Bu^(t) : t-butyl, Bzl: benzyl, OBzl: benzylester, OBu^(t) : t-butyl ester, OcHex: cyclohexylester, Br-Z:2-bromobenzyloxycarbonyl, 4CH₃ ·Bzl: 4-methylbenzyl, Dnp: dinitrophenyl,MBzl: methoxybenzyl, Cl₂ ·Bzl: 2,6-dichlorobenzyl,Mtr:4-methoxy-2,3,6-trimethylbenzenesulfonyl, Mts:mesitylene-2-sulfonyl, Acm: acetamidomethyl, Tos: p-toluenesulfonyl,Fmoc: 9-fluorenylmethyloxycarbonyl, NO₂ : nitro and HCO: formyl.

3. REAGENTS

DCC: dicyclohexylcarbodiimide, HOBt: 1-hydroxybenzotriazole, DTT:dithiothreitol, DCM: dichloromethane, DMF: dimethylformamide, MeOH:methanol, DIEA: diisopropylethylamine, THF: tetrahydrofuran, TFA:trifluoroacetic acid, HF: hydrogen fluoride, CH₃ CN: acetonitrile, NMP:N-methylpyrrolidone and DMSO: dimethyl sulfoxide.

SUMMARY OF THE INVENTION

The present inventors have found a novel γ-carboxyglutamic acidderivative represented by the formula (XI) shown below which can beutilized for introduction of γ-carboxyglutamic acid in the peptidesynthetic method. ##STR4## wherein n represents 0, 1 or 2.

The present invention also provides a method for preparing theγ-carboxyglutamic acid derivative represented by the formula (XI) and anoptically active derivative thereof wherein its stereostructure at theside chain is L and an intermediate for producing said derivative.

The present inventors have also conducted intensive investigations intoa method for synthesizing Gla⁷ human osteocalcin by introducing Gla atthe 17-position, the 21-position and the 24-position and have alsoprepared Glu⁷ human osteocalcin according to chemical synthesis byintroducing Gla at the 21-position and the 24-position, and consequentlyhave accomplished the present invention.

The present invention is a method for preparing Gla⁷ human osteocalcinand Glu⁷ human osteocalcin or salts of these, which comprisesintroducing γ-carboxyglutamic acid in a peptide synthetic method ofhuman osteocalcin by use of a protected optically active isomer (Lisomer) of γ-carboxyglutamic acid represented by the above formula (XI)or a salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing the ¹ H-NMR spectrum of the optically activeisomer (L isomer) of the compound (XI) obtained in Example 1;

FIG. 2 is a chart showing its ¹³ C-NMR spectrum;

FIG. 3 is a chart showing its IR-absorption spectrum;

and FIG. 4 is a BGP standard curve prepared based on the measuredresults shown in Table 3.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Peptide synthesis can be performed by the liquid phase method or thesolid phase method according to the methods described in, for example,Haruaki Yajima, Shunpei Sakakibara, edited by Biochemical Society ofJapan, Course of Biological Experiments (I), "Chemistry of Proteins"vol. 4, published by Tokyo Kagaku Dojin (1977); and Nobuo Izumiya et al"Basis and Experiment of Peptide Synthesis", published by Maruzen K. K.(1985). As the synthetic method of the human osteocalcin of the presentinvention, the solid phase method is preferred.

In the following, the case of synthesizing the Gla⁷ human osteocalcinand a salt thereof of the present invention according to the solid phasemethod is to be described.

First, the C-terminal amino acid of the desired human osteocalcin,namely Val is bound to an insoluble resin as the protected amino acid.The protected amino acid resin having such C-terminal protected aminoacid bound to an insoluble resin is commercially available and can beused as such. Subsequently, following the amino acid sequence of thehuman osteocalcin, protected amino acids are successively bonded fromthe C-terminal side to obtain a protected peptide resin. As theinsoluble resin, there may be employed any of those known in the fieldof art concerned, as exemplified by a chloromethyl resin, an oxymethylresin, a 4-(oxymethyl)phenylacetamidomethyl resin, and these resins canbe eliminatable with HF (hereinafter called "Pam" resin), etc.

The "protected amino acid" is an amino acid with its functional groupbeing protected according to a known method, and various kinds ofprotected amino acids are commercially available. When synthesizing thehuman osteocalcin of the present invention, it is preferred to chooseeither one of the protected amino acids shown below. First, theprotective group of α-amino group of amino acid is Boc or Fmoc. Theprotective group of guanidino group of Arg is Tos, NO₂, Mtr. Theprotective group of carboxyl group of Asp, Glu is Obzl, OBu^(t), OcHex.The protective group of mercapto group of Cys is 4CH₃ ·Bzl, MBzl, Acm.The protective group of imidazolyl group of His is Tos, Dnp, Fmoc. Theindolyl protective group of Trp is HCO or it may be also not protected.The protective group of hydroxyl group of Tyr is Br-Z, Cl₂ ·Bzl, Bzl,Bu^(t), or it may be also not protected.

The respective protective groups are required to be chosen adequatelydepending on the synthetic conditions of the peptide.

Gla at the 17-position, the 21-position and the 24-position in the Gla⁷human osteocalcin and Gla at the 21-position and the 24-position in theGlu⁷ human osteocalcin can be introduced as the protected Gla of theabove formula (XI). Synthesis of this protected Gla can be carried outaccording to the following method. ##STR5## That is, first the reactionbetween N-t-butyloxycarbonylserine (IV) and a benzyl halide is carriedout to synthesize a benzyl ester (V) (step a), then this is reacted withp-toluenesulfonyl chloride to form a sulfonate derivative (VI) (step b),which is reacted with a base such as diethylamine, etc. to synthesizeN-t-butyloxycarbonyldehydroalaninebenzyl ester (VII) (step c).

On the other hand, by transesterification of dimethyl malonate (VIII),dicycloalkyl malonate (IX) is formed (step d). The compound (IX) isreacted with the compound (VII) obtained in the step c in the presenceof sodium hydride as the base to synthesize a compound (X) (step e).Further, this is reduced to prepare a desired γ-carboxyglutamic acidderivative (XI) (step f). If desired, the compound (XI) obtained isoptically resolved according to the diastereomer salt method by use anoptical resolution agent such as quinine to prepare an optically activeisomer (L isomer) (II) (step g).

The above step a can be practiced by adding benzylbromide and a basesuch as triethylamine, etc. into an acetone solution oft-butyloxycarbonylserine (IV) and heating the mixture at a temperatureof room temperature to 100° C., preferably 40° C. to 70° C. for 5 hoursor longer, preferably 20 to 60 hours. After completion of the reaction,the solvent is evaporated and the residue is poured into water, theproduct is extracted with ether and the solvent evaporated under reducedpressure to give the compound (V).

The step b can be practiced by dissolving the compound (V) obtained inthe step a in a basic solvent such as pyridine, etc., addingp-toluenesulfonyl chloride to the solution and stirring the mixture at atemperature of -20° C. to 40° C., preferably -10° C. to 10° C. for 0.5to 20 hours, preferably 2 to 8 hours. After completion of the reaction,the reaction mixture is added to cold water, followed by stirring, andthe crystals formed are collected by filtration to give the compound(VI). Here, instead of the basic solvent, a base and a solvent such astriethylamine and THF, etc. can be used.

The step c can be practiced by dissolving the compound (VI) obtained ina mixed solution of ethyl acetate/diethyl ether, adding a base such asdiethylamine, etc. to the solution and stirring the mixture at atemperature of -20° C. to 40° C., preferably 0° C. to 30° C., for 0.5 to20 hours, preferably 2 to 6 hours. After completion of the reaction, thereaction mixture is filtered and the solvent evaporated under reducedpressure to give the compound (VII).

The step d can be practiced by adding cycloalkanol and a catalyst suchas p-toluenesulfonic acid into a toluene solution of dimethyl malonate(VIII) and stirring the mixture under heating at a temperature of 50° to200° C., preferably 90° to 120° C., while evaporating methanol formed,for 5 hours or longer, preferably 20 to 60 hours. After completion ofthe reaction, the product is washed with a sodium hydrogen carbonatesolution, the solvent evaporated under reduced pressure and then theresidue is distilled to give the compound (IX).

The step e can be practiced by adding the compound (IX) into asuspension of sodium hydride in a solvent such as THF, stirring themixture at a temperature of -40° to 70° C., preferably -10° to 40° C.for 0.1 to 10 hours, preferably 0.5 to 2 hours, subsequently addingdropwise a solution of the compound (VII) dissolved in the same solvent,and stirring the mixture at a temperature of -40° to 70° C., preferably-10° to 25° C., for 0.1 to 20 hours, preferably 0.5 to 10 hours. As thesolvent which can be used here, in addition to THF, toluene, ethylether, dioxane, etc. can be included. After completion of the reaction,the compound (X) can be obtained according to conventional isolation andpurification method such as extraction, column chromatography, etc.

The step f can be practiced by hydrolysis of the compound (X) obtainedat a hydrogen pressure of 0.1 to 20 kg/cm², preferably 0.5 to 5 kg/cm²,and at a temperature of 0° to 80° C., preferably 10° to 40° C. for 0.1to 20 hours, preferably 0.5 to 10 hours, in the presence of a catalyst,in a solvent such as methanol or ethanol. As the reducing catalyst whichcan be used here, palladium-carbon, platinum-carbon, etc. can beincluded. After completion of the reaction, by separating the catalystand evaporating the solvent under reduced pressure, the compound (XI)can be obtained.

The step g can be practiced by dissolving the compound (XI) in ethylacetate, adding an optical resolution reagent to the solution and thenrepeating filtration and recrystallization with methanol. As the opticalresolution reagent which can be used here, there can be includedalkaloids such as quinine, brucine, cinchonidine, cinchonine, etc.;amines such as (R)-(+)-1-phenethylamine, (S)-(-)-1-phenethylamine,(R)-(+)-1-(1-naphthyl)ethylamine, (S)-(-)-1-(1-naphthyl)ethylamine,etc.; hydrazides such as tyrosine hydrazide, etc. After the reaction,the product is desalted and crystallized with n-hexane, followed byfiltration, to give the optically active isomer (L isomer) (II) of thecompound (XI).

The compound (XI) and its optically active isomer (II) thus obtainedform salts with various inorganic bases and organic bases.

Examples of such salts can include alkali metal salts such as sodiumsalt, potassium salt, lithium salt; alkaline earth metals such ascalcium salt, magnesium salt; organic base salts such as of methylamine,ethylamine, diethylamine, triethylamine, benzylamine, pyrrolidine,piperidine, cyclohexylamine, dicyclohexylamine, ethanolamine,diethanolamine, ornithine, lysine, arginine.

The above compound can be identified by its IR absorption, NMR spectrumand mass analysis. The optical purity of the optically active isomer canbe determined by forming a diastereomer with an optically active basesuch as (+)-naphthylamine, (-)-naphthylethylamine, (+)-phenethylamine,(-)-phenethylamine, etc., separating this by conventional liquidchromatography and calculating the ratio of both substances. In thiscase, it is preferable to determine a correction coefficient accordingto the same operations also for the racemic mixture and correct theabove optical purity by use thereof.

The configuration of the optically active γ-carboxyglutamic acidderivative of the present invention can be determined by hydrolyzingthis substance in 0.1% phenol containing 6 N hydrochloric acid underreduced pressure at 110° C. for 22 hours to convert it to glutamic acid,and comparing the eluted position in high performance liquidchromatography of the reaction product of this with2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylisothiocyanate (hereinaftercalled GITC) according to the method of, for example, Futamura etal.[Journal of Chromatography, 316, 547-552 (1984)] with that of thereaction product of the standard L-glutamic acid, D-glutamic acid andGITC.

Protected amino acids can be bound according to conventionalcondensation methods such as the DCC method, the active ester method,the mixed or symmetric acid anhydride method, the carbonylimidazolemethod, the DCC-HOBt method, the diphenylphosphorylazide method, etc.,of which the DCC method, the DCC-HOBt method, the symmetric acidanhydride method are preferred. These condensation reactions are carriedout generally in an organic solvent such as DCM, DMF, NMP, chloroform,DMSO, benzene, etc. or a solvent mixture of them, but preferably in DCM,DMF or a solvent mixture of these. As the elimination reagent of theprotective group of α-amino group, TFA/DCM, HCl/dioxane, piperidine/DMF,etc. may be employed, and may be chosen suitably depending on the kindof said protective group. The extent of progress of the condensationreactions in the respective steps of synthesis may be examined by themethod of E. Kaiser et al.[Anal. Biochem., 34, 595 (1970)] (Ninhydrinreaction method), etc.

Protected peptide resins having desired amino acid sequences areobtained as described above, and specific examples thereof are shownbelow. ##STR6## The protected peptide resin (Boc-Val-Pam resin) can betreated with a reagent which can eliminate the peptide from the resinand further eliminate side chain protective groups of the respectiveamino acids, for example, HF, TFA, etc. (final deprotection reaction) togive a peptide with mercapto group of Cys being deprotected.

By oxidizing the peptide in a buffer to form an intramolecular disulfidebond, the peptide can be obtained. More specifically, by dissolving thepeptide with mercapto group of Cys being deprotected in a buffer to aconcentration of 10⁻³ to 10⁻⁷ mole/liter, preferably 10⁻⁴ to 10⁻⁵mole/liter, adjusting pH to 6.0 to 8.5, preferably 7.0 to 8.0, and thenstirring the solution at 4° to 50° C., preferably 4° C. to roomtemperature for 4 to 30 hours, a crude human osteocalcin can beobtained. The buffer to be used in this reaction is known and mayinclude, for example, ammonium acetate, Tris.HCl, etc. Also, as thepromoting agent of this reaction, ferricyanate (potassium ferricyanate,etc.) can be added.

The crude human osteocalcin thus obtained can be purified by isolationby extraction, recrystallization, various chromatographic techniques(gel filtration, ion exchange, partition, adsorption, reverse phase),electrophoresis, countercurrent partition, etc., but the methodaccording to reverse phase high performance liquid chromatography(reverse phase HPLC) is the most effective.

By utilizing human BGP (Gla¹⁷ or Glu¹⁷ human osteocalcin) according tothe present invention, it can be realized to develop an immunologicalassay method of human BGP, and by effecting measurement of BGP incirculating blood, information for grasping condition of illnessconcerning bone metabolism can be obtained. That is, measurement of BGPin circulating blood of human has been carried out by an immunologicalassay method such as the radioimmunoassay (RIA) and the enzymeimmunoassay (EIA) which utilize antiserum or a monoclonal antibody tobovine BGP or human BGP obtained from immunized rabbit, mouse or goat,but since a cross reaction with bovine BGP is utilized or a specificantibody is utilized for a fragment, these methods involve problems inmeasuring specificity, particularly measuring only intact molecule of 1to 49, or measuring time and sensitivity should be improved.

Thus, an immunoradiometric assay (IRMA) which is specific to human BGP(1 to 49) can be accomplished by utilizing two monoclonal antibodies, inwhich one antibody is an anti-human BGP monoclonal antibody which isobtained by using the human BGP (Gla¹⁷ and Glu¹⁷ human osteocalcin) ofthe present invention as antigen and is specific to amino acidalignments at (12 to 33) of human BGP and the other antibody is ananti-bovine BGP monoclonal antibody which is specific to amino acidalignments at (30 to 49) of human BGP.

PREPARATION OF MONOCLONAL ANTIBODY PRODUCING HYBRIDOMA Step aPREPARATION OF ANTI-HUMAN BGP MONOCLONAL ANTIBODY PRODUCING HYBRIDOMA

The human BGP is a polypeptide comprising 49 amino acid residues andrelatively low molecular weight, and thus it does not have sufficientimmunogenicity. Accordingly, after it is combined with key-hole limpethemocyanine, bovine serum albumin or swine thyroglobulin to prepare acomposite material, the composite material is emulsified with a suitableimmunoactivator such as a Freund's complete adjuvant to prepare anemulsion and the emulsion is injected into a mouse. The mouse to be usedis generally a Balb/c species but any kind of mice which is normallyused for immuniozation purposes can be used. Immunization can be carriedout by repeatedly inoculating the abdomen of a mouse with the aboveemulsion by administration hypodermically several times for each severalweeks. After 3 to 5 days from final immunization, the spleen is removed,and the spleen cells are adjusted and fused with a mouse myeloma cell(e,g, P3-X63-Ag8-U1, NS-1, X63-Ag8.653) to prepare a hybridoma. Cellfusion is carried out by the PEG method and screening of the fusedstrain is due to HAT selective medium. Screening for antibody productionof the hybridoma is carried out by a known method such as EIA, RIA andthe membrane fluorescent antibody technique whereby the hybridomasdesired, which generate immunoglobulin specific to human BGP, areselected. Cloning of the hybridoma is carried out by the known methodsuch as the limiting dilution method, or a method wherein whileobserving with a microscope, catching single cell with a glasscapillary, etc.

Step b PREPARATION ANTI-BOVINE MONOCLONAL ANTIBODY PRODUCING HYBRIDOMA

It can be carried out in accordance with the preparation of themonoclonal antibody producing hybridoma to human BGP.

Production of monoclonal antibody

Production of monoclonal antibody from the resulting hybridoma becarried out by two methods one of which is an in vitro system whereinthe antibody is produced as a cultivated supernatant in a cultivatingapparatus and the other of which is an in vivo wherein it is produced inthe form of ascites in animal bodies such as abdominal cavity of mouse.In the in vitro system, the medium can be used by adding a suitableamount of bovine fetal serum to a usual Eagle's MEM, RPMI-1640 orDulbecco's modified Eagle's medium. Cultivation period is generally 3 to7 days. In the in vitro cultivation system, after inoculating hybridomastrains in abdominal cavity of mammal such as mouse, ascites iscollected between 4 and 14 days to obtain monoclonal antibody asascites. A large amount of monoclonal antibody can be obtained generallyby the in vivo system.

The monoclonal antibody (ascites or cultivation supernatant) obtained bythe above methods is subjected to purification by combining knownmethods such as salting out, DEAE-cellulose column chromatography orprotein A-Sepharose column chromatography, etc.

The present inventors have established the anti-human BGP monoclonalantibody (MBG 04F5) and anti-bovine BGP monoclonal antibody (MBG 14AX)and examined their characteristics. As a result, it has been found thatMBG 04F5 recognizes N-terminal to intermediate portion of human BGPsince it strongly recognizes the human BGP (12 to 33) and does not reactwith N-terminal and C-terminal fragments. It has been also shown thatMBG 14AX recognizes C-terminal side since it strongly recognizes thehuman BGP (30 to 49) and does not react with N-terminal to intermediateportion thereof.

As the immunological assay method of human BGP using the abovemonoclonal antibodies MBG 14AX and MBG 04F5, there may be mentioned RIAwhich is a second antibody method, the sandwich type EIA methods, thesandwich type fluorescent immuno-assay (FIA) method and the sandwichtype IRMA method. With respect to the IRMA method, as shown in Referenceexamples, the one step IRMA method may be mentioned wherein a sample ora standard BGP, a predetermined amount of monoclonal antibody MBG 14AXwhich is radioactive isotope labelled, and polystyrene beads covered bymonoclonal antibody MBG 04F5 are reacted, and then the dose ofradioactivity bound to the beads is measured. During this time, thesystem wherein MBG 14AX is immobilized on polystyrene beads and MBG 04F5is radioactive isotope labelled are also possible. Further, theso-called two step IRMA wherein a sample or a standard BGP is firstreacted with antibody-covered beads, and then after removing thereaction solution, a monoclonal antibody which is radioactive isotopelabelled is reacted thereto is also possible. Moreover, the reversereaction IRMA wherein a sample or a standard BGP is first reacted with amonoclonal antibody which is isotope labelled, and then reacted withantibody-coated beads is also possible.

At this time, as the carrier to immobilize the antibody, there may beutilized a carrier for antigen-antibody which is used for generalimmunoassay such as particulates made of a glass, magnetic material orplastics, or spherical materials (beads), tube and plate. To thesecarriers was immobilized an antibody which recognizes N-terminal,intermediate portion or C-terminal of the human BGP physically or by thecovalent bonding. The antibody to be used at this time may by either themonoclonal antibody or the polyclonal antibody since they can be treatedin the same manner.

Radioactive isotope labelling of an antibody which recognizesN-terminal, intermediate portion or C-terminal of the human BGP can becarried out by incorporating an radioactive isotope such as ¹²⁵ I by theknown methods such as Chloramine-T method, lactoperoxidase method,Iodogen method and Bolton-Hunter method.

The RIA due to the second antibody method is possible either themomoclonal antibody or polyclonal antibody. The method may include twomethods one of which is a competitive method wherein a sample or astandard BGP, an anti-BGP antibody and a human BGP which is radioactiveisotope labelled are simultaneously reacted, B/F separation is carriedout by a second antibody and measuring a dose of radioactivity bound tothe antibody, and the other is a non-equilibrium method wherein additionof a human BGP which is radioactive isotope labelled is delayed forseveral hours to 1 day.

The sandwich EIA and the sandwich FIA are basically the same as the IRMAbut different therefrom in using an enzyme label or a fluorescentsubstance label in place of an isotope label.

An antibody for labelling may be used by purifying until IgG and it isused as a labelling substance as it were, or used by digesting withpepsin to F(ab')₂ or reducing with 2-mercaptoethanol to Fab'.

As the enzyme for labelling, alkaline phosphatase, β-D-galactosidase,peroxidase and glucose oxidase can be used.

As the fluorescent labelling, a fluorescent substance such asfluorescein, fluorescein isocyanate, fluorescein isothiocyanate andrhodamine may be used; and it is also possible to use a method in whichan europium ion is used as a label.

In the one step IRMA shown in Reference example, it has been shown thatonly the intact BGP (1 to 49) is measured so that the problem ofspecificity in the conventional RIA can be overcome. Therefore, it issuggested that it is a measuring system capable of providing availabledata for clinically evaluating bone metabolism.

EXAMPLES

The present invention is described in detail below by referring toExamples, but the present invention is not limited by these Examples atall.

EXAMPLE 1 Synthesis of γ-carboxyglutamic acid derivative Step aSYNTHESIS OF COMPOUND (V)

Into 200 ml of an acetone solution of 25 g of N-t-butyloxycarbonylserine(IV) were added 26.8 g of benzylbromide and 16.9 g of triethylamine, andthe mixture was heated under reflux with stirring for 2 days. Aftercompletion of the reaction, the reaction mixture was concentrated underreduced pressure to evaporate the solvent, and water and diethyl etherwere added to the residue, followed by thorough shaking and liquidseparation. The ether was washed twice with water, subsequently oncewith saturated aqueous sodium chloride. The ether layer was separated,dried and then ether was evaporated under reduced pressure to give 30.3g of an oily compound (V) (yield 97.8%).

Step b SYNTHESIS OF COMPOUND (VI)

Into a solution of 31 g of the compound (V) obtained in the step adissolved in 100 ml of pyridine was added 23 g of p-toluenesulfonylchloride, and after stirring at 0° C. for 3 hours, 2.3 g ofp-toluenesulfonyl chloride was further added, followed further bystirring for 4 hours. After completion of the reaction, the reactionmixture was poured into ice-water, the mixture was stirred, and thewhite crystals formed were collected by filtration to give 37.4 g of thecompound (VI) (yield: 79.3%).

Step c SYNTHESIS OF COMPOUND (VII)

Into a solution of 37.4 g of the compound (VI) obtained in the step bdissolved in 200 ml of a mixed solution of ethyl acetate/diethyl ether(1:1) was gradually added dropwise 17.2 ml of diethylamine. and themixture was stirred at room temperature for 3 hours. After completion ofthe reaction, the reaction mixture was cooled to 0° C., filtered and thefiltrate was subjected to evaporation under reduced pressure to give23.0 g of an oily compound (VII) (yield: 99.3%).

Step d SYNTHESIS OF COMPOUND (IX)

Into 250 ml of a toluene solution of 33 g of dimethyl malonate (VIII)were added 75 g of cyclohexanol and 1.25 g of p-toluenesulfonic acidmonohydrate, and the mixture was heated under reflux with stirring for42 hours. After completion of the reaction, the reaction mixture waswashed twice with an aqueous sodium hydrogen carbonate, twice withwater, and further twice with saturated aqueous sodium chloride. Theorganic layer was dried over anhydrous sodium sulfate, and the solventwas evaporated under reduced pressure. The residue was distilled, andthe distillates at 128° to 130° C. were collected by separation to give49.7 g of an oily compound (IX) (yield: 74.2%).

Step e SYNTHESIS OF COMPOUND (X)

Into 150 ml of a THF suspension of 3.32 g of 60% sodium hydride inmineral oil was gradually added dropwise a solution of 22.8 g of thecompound (IX) obtained in the step d dissolved in THF under cooling to5° C. After foaming ceased, the mixture was stirred at room temperaturefor one hour. Next, a solution of 23 g of the compound (VII) obtained inthe step c dissolved in THF was added dropwise into the previousreaction mixture under ice-cooling, and the mixture was stirred for onehour. After completion of the reaction, the reaction mixture was mixedwith 0.1 N citric acid under ice-cooling and the mixture extracted withdiethyl ether. The ether layer was washed with water, then withsaturated aqueous sodium chloride, and after drying, the solvent wasevaporated under reduced pressure. To the residue were addedacetonitrile and n-hexane to effect extraction, and the acetonitrilelayer was separated, followed by evaporation of the solvent underreduced pressure. The residue was purified by silica gel columnchromatography with diethyl ether/n-hexane (2:5) to give 32.7 g of anoily compound (X) (yield: 72.5%).

Step f SYNTHESIS OF COMPOUND (XI)

Into a solution of 28.8 g of the compound (X) obtained in the step edissolved in 300 ml of ethanol was added 1.5 g of 5% palladium-carbon,and the mixture was stirred in hydrogen gas atmosphere at roomtemperature for 3 hours. After completion of the reaction, the catalystwas removed and ethanol was evaporated under reduced pressure to give23.0 g of an oily compound (XI) (yield: 95.7%).

F_(ab) mass analysis [M+H]⁺ Found: 456, Calcd.: 456.

Step g OPTICAL RESOLUTION OF COMPOUND (XI)

The compound (XI) obtained in the step f (23.0 g) was dissolved in 100ml of ethyl acetate, 200 ml of an ethyl acetate solution of 17.1 g ofquinine was added to the solution and the mixture was shaken and thenleft to stand at room temperature for 3 days. The crystals formed werecollected by filtration to obtain 21.8 g of an L-γ-carboxyglutamic acidderivative quinine salt with an optical purity of 39.2% as white needles(yield: 53.0%). This was further recrystallized three times repeatedlywith methanol, followed by suspending in ethyl acetate and desaltingwith 1 N citric acid. The organic layer was washed with saturatedaqueous sodium chloride, dried, and then ethyl acetate was evaporatedunder reduced pressure. To the residue was added n-hexane, and whitecrystals formed were collected by filtration to give 4.79 g of anL-γ-carboxyglutamic acid derivative (II) with an optical purity of 97.2%(yield: 20.6%).

Nuclear magnetic resonance spectra of the optically active isomers (II)obtained are shown in FIG. 1 and FIG. 2, and the IR-absorption spectrumin FIG. 3.

Elemental analysis (as C₂₃ H₃₇ NO₈)

    ______________________________________                                                C           H      N                                                  ______________________________________                                        Found:    60.70         8.42   3.00                                           Calcd.:   60.64         8.19   3.08                                           ______________________________________                                    

F_(ab) mass analysis [M+H]⁺ Found: 456, Calcd.: 456.

Optical rotation [α]_(D) : -4.9 ° (C 1.05, methanol).

EXPERIMENTAL EXAMPLE 1 OPTICAL PURITY ASSAYING METHOD

The quinine salt of the optically active isomer (II) obtained in theabove Example 1 (46.8 mg) was suspended in ethyl acetate and desaltedwith 1 N citric acid. The organic layer was washed with saturatedaqueous sodium chloride and dried. Evaporation of ethyl acetate underreduced pressure gave 27.5 mg of a free γ-carboxyglutamic acidderivative (hereinafter called sample (a)). Its whole amount wasdissolved in 2.8 ml of dichloromethane and to the resultant solutionwere added 14.2 mg of 2-chloro-1-methylpyridinium, 8.2 μl ofR-(+)-1-(α-naphthyl)ethylamine and 26.6 μl of n-butylamine, and thereaction was carried out at 40° C. for 2 hours. After completion of thereaction, diastereomer fractions formed by thin layer chromatographywere separated. The fractions were eluted with ethyl acetate, and thenthe residue obtained by evaporation under reduced pressure was analyzedby high performance liquid chromatography under the followingconditions.

ANALYTICAL CONDITIONS

Column: Ricrosolve Si60, 5 μm φ4.6×250 mm (Merck).

Moving phase: ethyl acetate/n-hexane=1 : 3.

Flow rate: 1 ml/min.

Detection: UV 254 nm.

Column temperature: 30° C.

Data processing device: Chromatopack C-R3A (Shimazu Seisakusho).

Also for 11.2 mg of the racemic mixture (XI) of the above compound,diastereomers with 3.3 μl of R-(+)-1-(α-naphthyl)ethylamine weresimilarly synthesized, and analyzed by high performance liquidchromatography under the same conditions as mentioned above. The resultsare shown in the following Table.

    ______________________________________                                        Diastereomer      Peak area                                                   acyl components   L*.sup.1                                                                              D*.sup.1                                            ______________________________________                                        Sample (a)        284,147  4,396                                              Racemic mixture   207,611 222,716                                             ______________________________________                                         *.sup.1 (Configuration of diastereomer acyl component side chain.             Determined by Experimental example 2.)                                   

The optical purity was determined by the following formula.

    Optical purity (%)=(2×-1)×100,

wherein ##EQU1## The optical purity of the above optically active isomer(L isomer) (II) was found to be 97.2%.

EXPERIMENTAL EXAMPLE 2 DETERMINATION OF OPTICAL ISOMERS

One mg of the above optically active isomer (II) was hydrolyzed with0.1% phenol containing 6 N hydrochloric acid under reduced pressure at110° C. for 22 hours. After completion of the reaction, hydrochloricacid was volatilized and the residue was dissolved in 5 ml of 0.1 Nhydrochloric acid. To 30 μl of the solution were added 30 μl of 50 mg/mlof triethylamine acetonitrile solution and 60 μl of 10 mg/ml of GITCacetonitrile solution, and the reaction was carried out at roomtemperature for 20 minutes. The same procedures were conducted also for1 mg of the standard L-glutamic acid and D-glutamic acid and blank. Therespective solutions obtained were analyzed by reverse phase HPLC.

ANALYTICAL CONDITIONS

Column: YMC-R, 5 μm φ4.6×250 mm (Yamamura Kagaku).

Moving phase: A - 0.1% phosphoric acid, B - acetonitrile.

Concentration gradient: A/B=100/10→50/50, eluted according to a linearconcentration gradient for 30 minutes.

Detection: UV 250 nm.

Column temperature: room temperature.

Data processing device: Chromatopack C-R3A (Shimazu Seisakusho).

Analysis was conducted in the order of L-glutamic acid reaction mixture,D-glutamic acid reaction mixture, optically active isomer (II) reactionmixture, D-glutamic acid reaction mixture+optically active isomer (II)reaction mixture and blank. The relative retention times of thediastereomers formed on the chromatogram were L-glutamic aciddiastereomer<D-glutamic acid diastereomer, and the optically activediastereomer was found to be enriched in L-glutamic acid.

From the above results, the optically active isomer (II) was identifiedto be the L-isomer.

EXAMPLE 2 SYNTHESIS OF GLA¹⁷ HUMAN OSTEOCALCIN REPRESENTED BY THEFORMULA ##STR7## (1) INTRODUCTION OF 48-POSITION PRO INTO BOC-VAL-PAMRESIN 1) DEPROTECTION AND NEUTRALIZATION

An amount 0.746 g of Boc-Val-Pam resin (0.67 mmole/g) was washed twicewith DCM. To the resin was added 8 ml of a 33 % TFA solution (solvent:DCM), and after stirring for 80 seconds, the mixture was filtered.Further, 80 ml of a 50 % TFA solution (solvent: DCM) was added, andafter stirring for 18.5 minutes, the mixture was subjected to filtrationto eliminate Boc groups. The resin obtained was consecutively treatedwith the solvents shown below, and filtration effected after eachtreatment.

DCM (×3, each for 30 seconds).

10% DIEA/DMF (×2, each for one minute).

DMF (×5, each for 30 seconds).

2) PREPARATION OF BOC-PRO SYMMETRIC ACID ANHYDRIDE

After 2 mmole of Boc-Pro was dissolved in 3 ml of DCM, 2 ml of a 0.5 MDCC solution (solvent: DCM) was added thereto to carry out the reactionfor 8 minutes, thereby forming symmetric acid anhydride. After removalof dicyclohexylurea by-produced by filtration, 4 ml of DMF was added,followed subsequently by evaporation of DCM in the reaction mixture.

3) CONDENSATION REACTION

The DMF solution of the Boc-Pro symmetric acid anhydride prepared in 2)was added to the Val-Pam resin prepared in 1) and the reaction wascarried out at room temperature for 18 minutes. After completion of thereaction, the reaction mixture was washed with DCM for 5 times.

(2) INTRODUCTION OF THE RESPECTIVE AMINO ACIDS AT 47- TO 1-POSITIONS

Similarly as described in (1), Boc-Pro-Val-Pam resin was successivelycoupled with the protective amino acids corresponding to the respectiveconstituent amino acids from the 47-position to the 1-position of Gla¹⁷human osteocalcin. Table 1 shows the protected amino acids, syntheticmethods, etc. used in the respective reaction steps.

In the Table, concerning the synthetic cycle, the method (1), the method(2), the method (3) mean deprotection, neutralization and condensationreactions carried out according to the following procedures,respectively.

Method (1)

1) Deprotection with 33% TFA (solvent: DCM) for 80 seconds;

2) Deprotection with 50% TFA (solvent: DCM) for 18.5 minutes;

3) DCM washing, ×3, each for 30 seconds;

4) Neutralization with 10% DIEA/DMF, ×2, each for 1 minute;

5) DMF washing, ×5, each for 30 seconds;

6) Condensation reaction for 18 minutes;

7) DCM washing, ×5, each for 30 seconds.

Method (2)

1)-5): the same as in Method (1);

6) Condensation reaction for 26 minutes;

7) the same as in Method (1).

Method (3)

1)-5): the same as in Method (1);

6) Condensation reaction for 42 minutes;

7) DMF washing, ×3, each for 30 seconds;

8) Neutralization with 10% DIEA/DMF for 45 seconds;

9) DMF washing for 30 seconds;

10) DCM washing for 3 times, each for 30 seconds;

11) Condensation reaction for 42 minutes;

12) DMF washing for 30 seconds;

13) DCM washing, ×5, each for 30 seconds.

Also, in the Table, concerning synthesis of symmetric acid anhydride orHOBt ester, Method (1), Method (2), Method (3), Method (4) and Method(5) mean syntheses of symmetric acid anhydride or HOBt ester accordingto the following procedures, respectively.

Method (1)

After 2 mmole of Boc-amino acid was dissolved in 3 ml of DCM, 2 ml of0.5 M DCC solution (solvent DCM) was added thereto, the reaction wascarried out for 8 minutes to form a symmetric acid anhydride. Afterremoval of dicyclohexylurea by-produced by filtration, 4 ml of DMF wasadded, and subsequently DCM in the reaction mixture was evaporated.

Method (2)

After 2 mmole of Boc-amino acid was dissolved in 3 ml of DCM, 2 ml of0.5 M DCC solution (solvent: DCM) was added thereto, and the reactionwas carried out for 8 minutes to form a symmetric acid anhydride. Afterremoval of dicyclohexylurea by-produced by filtration, 1 ml of DMF wasadded and the DCM in the reaction mixture was evaporated.

Method (3)

After 2 mmole of Boc-amino acid was dissolved in 0.3 ml of DMF and 2.5ml of DCM, 2 ml of 0.5 M DCC solution (solvent: DCM) was added thereto,and the reaction was carried out for 8 minutes to form a symmetric acidanhydride. After dicyclohexylurea by-produced was removed by filtration,4 ml of DMF was added and subsequently DCM in the reaction mixture wasevaporated.

Method (4)

After 4 ml of 0.5 M HOBt and 0.3 ml of DCM were added into 2 mmole ofBoc-amino acid to be dissolved therein, 4 ml of 0.5 M DCC solution(solvent: DCM) was added to the solution, and the reaction was carriedout for 33 minutes to form an HOBt ester. After dicyclohexylureaby-produced was removed by filtration, 3 ml of DCM in the reaction wasevaporated. Preparation of the HOBt ester according to this operationwere practiced twice for one residue.

Method (5)

After 4 ml of 0.5 M HOBt and 1.5 ml of DCM were added to 2 mmole ofBoc-amino acid to be dissolved therein, 4 ml of 0.5 M DCC solution(solvent: DCM) was added to the solution, and the reaction was carriedout to form an HOBt ester. After removal of dicyclohexylurea by-producedby filtration, 3 ml of DCM in the reaction mixture was evaporated.Preparation of the HOBt ester according to this operation was performedtwice for one residue.

After introduction of the amino acid at the 1-position, 20 ml of a 33%TFA solution (solvent: DCM) was added to the resin peptide, and themixture was stirred for 80 seconds and then filtered. Further, 20 ml ofa 50% TFA solution (solvent: DCM) was added, and the mixture was stirredfor 18.5 minutes and filtered to eliminate Boc groups. The resinobtained was successively treated with the following solvents, andfiltered after each treatment:

DCM (×3, each for 30 seconds);

10% DIEA/DCM (×2, each for one minute);

DCM (×5, each for 30 seconds).

Next, the present resin peptide was dried under reduced pressure for oneday and night to obtain a dry resin peptide.

                  TABLE 1                                                         ______________________________________                                                                       Synthesis of                                   Position                       symmetric                                                                              Cou-                                  of                             acid     pling                                 amino  Protected     Synthesis anhydride or                                                                           num-                                  acid   amino acid    cycle     HOBt ester                                                                             ber                                   ______________________________________                                        47     Bos--Gly      Method (1)                                                                              Method (2)                                                                             1                                     46     Bos--Tyr(Br--Z)                                                                             Method (1)                                                                              Method (1)                                                                             1                                     45     Bos--Phe      Method (2)                                                                              Method (1)                                                                             1                                     44     Bos--Arg(Tos) Method (3)                                                                              Method (5)                                                                             2                                     43     Bos--Arg(Tos) Method (3)                                                                              Method (5)                                                                             2                                     42     Bos--Tyr(Br--Z)                                                                             Method (1)                                                                              Method (1)                                                                             1                                     41     Bos--Ala      Method (1)                                                                              Method (1)                                                                             1                                     40     Bos--Glu(OBzl)                                                                              Method (1)                                                                              Method (1)                                                                             1                                     39     Bos--Gln      Method (3)                                                                              Method (4)                                                                             2                                     38     Bos--Phe      Method (2)                                                                              Method (1)                                                                             1                                     37     Bos--Gly      Method (1)                                                                              Method (2)                                                                             1                                     36     Bos--Ile      Method (2)                                                                              Method (1)                                                                             1                                     35     Bos--His(Tos) Method (2)                                                                              Method (2)                                                                             1                                     34     Bos--Asp(OBzl)                                                                              Method (1)                                                                              Method (1)                                                                             1                                     33     Bos--Ala      Method (1)                                                                              Method (1)                                                                             1                                     32     Bos--Leu      Method (2)                                                                              Method (3)                                                                             1                                     31     Bos--Glu(OBzl)                                                                              Method (1)                                                                              Method (1)                                                                             1                                     30     Bos--Asp(OBzl)                                                                              Method (1)                                                                              Method (1)                                                                             1                                     29     Bos--Cys(4CH.sub.3 Bzl)                                                                     Method (2)                                                                              Method (1)                                                                             1                                     28     Bos--Asp(OBzl)                                                                              Method (1)                                                                              Method (1)                                                                             1                                     27     Bos--Pro      Method (2)                                                                              Method (1)                                                                             1                                     26     Bos--Asn      Method (3)                                                                              Method (4)                                                                             2                                     25     Bos--Leu      Method (2)                                                                              Method (3)                                                                             1                                     24     Bos--Gla(OcHex).sub.2                                                                       Method (1)                                                                              Method (1)                                                                             1                                     23     Bos--Cys(4CH.sub.3 Bzl)                                                                     Method (2)                                                                              Method (1)                                                                             1                                     22     Bos--Val      Method (2)                                                                              Method (1)                                                                             1                                     21     Bos--Gly      Method (1)                                                                              Method (1)                                                                             1                                     20     Bos--Gly      Method (3)                                                                              Method (5)                                                                             2                                     19     Bos--Arg(Tos) Method (3)                                                                              Method (5)                                                                             2                                     18     Bos--Pro      Method (2)                                                                              Method (1)                                                                             1                                     17     Bos--Gla(OcHex).sub.2                                                                       Method (1)                                                                              Method (1)                                                                             1                                     16     Bos--Leu      Method (2)                                                                              Method (3)                                                                             1                                     15     Bos--Pro      Method (2)                                                                              Method (1)                                                                             1                                     14     Bos--Asp(OBzl)                                                                              Method (1)                                                                              Method (1)                                                                             1                                     13     Bos--Pro      Method (2)                                                                              Method (1)                                                                             1                                     12     Bos--Tyr(Br--Z)                                                                             Method (2)                                                                              Method (1)                                                                             1                                     11     Bos--Pro      Method (2)                                                                              Method (1)                                                                             1                                     10     Bos--Val      Method (1)                                                                              Method (1)                                                                             1                                      9     Bos--Pro      Method (1)                                                                              Method (1)                                                                             1                                      8     Bos--Ala      Method (1)                                                                              Method (1)                                                                             1                                      7     Bos--Gly      Method (1)                                                                              Method (2)                                                                             1                                      6     Bos--Leu      Method (2)                                                                              Method (3)                                                                             1                                      5     Bos--Trp      Method (2)                                                                              Method (3)                                                                             1                                      4     Bos--Gln      Method (3)                                                                              Method (4)                                                                             2                                      3     Bos--Tyr(Br--Z)                                                                             Method (1)                                                                              Method (1)                                                                             1                                      2     Bos--Leu      Method (2)                                                                              Method (3)                                                                             1                                      1     Bos--Tyr(Br--Z)                                                                             Method (1)                                                                              Method (1)                                                                             1                                     ______________________________________                                    

(3) Decomposition with HF

A part (815 mg) of the dried resin peptide was weighed, placed in areactor (made of Teflon) for HF decomposition, 2 ml of anisole wasadded, and the mixture was left to stand overnight to swell the resin.The above reactor was a stirrer introduced therein was mounted on a HFdecomposition device (Peptide Research Institute), placed in a dryice-methanol bath, and 18 ml of HF was introduced into the reactor. Themixture was stirred in an ice bath at 0° C. for one hour. HF wasgradually evaporated under reduced pressure. After 3 hours, the reactorwas dismantled and the resin peptide decomposed product was taken outfrom the reactor by use of anhydrous diethylether and washed withanhydrous diethylether. The resin peptide decomposed product was addedinto 50 ml of 30% acetic acid to dissolve the deprotected peptide. Thiswas previously substituted to acetic acid form for the purpose of saltexchange and passed through a Dowex 1×2 ion exchange resin column. Tothe fractions passed as such were added water to adjust the acetic acidconcentration to 1 N, followed by lyophilization to obtain 475 mg ofreduced type crude Gla¹⁷ human osteocalcin.

(4) FORMATION OF DISULFIDE BOND BY AIR OXIDATION

Of the reduced type crude human osteocalcin obtained in (3), 287 mg wasdissolved in 0.1 M ammonium acetate solution (pH 8.5) and 100equivalents of DTT were added, followed by reduction at 40° C. for 5hours. By this reduction operation, the dimer which can be formed byoxidation of mercapto group is returned to the monomer.

The reaction mixture was adjusted to pH 4.0 by addition of acetic acidand then centrifuged. After removal of the supernatant, the precipitateswere redissolved by addition of 30% acetic acid. Subsequently, DTT andsalts were removed by gel chromatography by use of Sephadex G25(Pharmacia). Next, the fractions containing the reduced type crude Gla¹⁷human osteocalcin were gradually added dropwise into 0.1 M ammoniumacetate solution (pH 7.5) while maintaining pH constant. The peptideconcentration at this time was made about 1.5×10⁻⁶ M. After formation ofdisulfide bond by stirring at room temperature for 15 days, the reactionmixture was adsorbed onto a column filled with octadecylsilica (ODScolumn, φ2×30 cm), washed with 0.1% TFA, and then the peptide was elutedwith 60% acetonitrile solution. After evaporation of acetonitrile underreduced pressure, the residue was lyophilized to obtain crude Gla¹⁷human osteocalcin. The disulfide formation reaction was monitored by theknown Elman test by use of 5,5'-dithiobis(2-nitrobenzoic acid), and thereaction product finally obtained had an oxidation ratio of 95%.

(5) PURIFICATION OF GLA¹⁷ HUMAN OSTEOCALCIN BY REVERSE PHASE HPLC

The crude Gla¹⁷ human osteocalcin obtained in (4) was dissolved in 30%acetic acid (10 mg/ml) and purified by high performance liquidchromatography isocratic elution. The column employed was YMC-D (YMC,φ2×30 cm), and the eluants employed were A solution of water (100) - 10%TFA (1) and B solution of water (40) - acetonitrile (60) - 10% TFA (1),and elution was carried out under the conditions of A (48) - B (52).Here, the numerals within the brackets are volume ratios. The fractionscorresponding to Gla¹⁷ human osteocalcin were separated and lyophilizedto give white powder.

Further, the above white powder was dissolved in 30% acetic acid (10mg/ml), and repurified similarly as described above. As the eluant,A/B=51/49 was employed. The fractions corresponding to Gla¹⁷ humanosteocalcin were separated and lyophilized to give white powder.

The white powder obtained by repurification was further dissolved in 30%acetic acid (10 mg/ml), and purification was further carried out underthe same conditions as in repurification. Fractions corresponding toGla¹⁷ human osteocalcin were collected and lyophilized to obtain whitepowder. This product was dissolved in 30% acetic acid, desalted bySephadex G25 column chromatography, then water was added to the Gla¹⁷human osteocalcin fractions to control the acetic acid concentration to1 N, followed by lyophilization, to give 6.6 mg of the desired Gla¹⁷human osteocalcin.

(6) STRUCTURE IDENTIFICATION AND PURITY ASSAY OF PURIFIED GLA¹⁷ HUMANOSTEOCALCIN

Analytical values of amino acids of the purified Gla¹⁷ human osteocalcinare shown in Table 2.

This product (100 μg) was dissolved in 1% NH₄ HCO₃, and digested at 25°C. for 2 hours with addition of 1/10 equivalent of trypsin-TPCK(Worsinton). As the result of measurement of the digested product byreverse phase HPLC under the following measuring conditions (1), threemain peaks were recognized at 18.9 min., 47.6 min and 66.8 min. on thechromatogram, and from the results of amino acid analysis and Fab massanalysis, they were found to be the peptides corresponding to the 45-49positions, 20-43 position and 1-19 positions of Gla¹⁷ human osteocalcin,respectively.

                  TABLE 2                                                         ______________________________________                                        Amino acid composition of purified Gla.sup.17 human osteocalcin               Amino acid  Molar ratio                                                                             Amino acid number                                       ______________________________________                                        Gla*        2.93      3                                                       Asx**       5.23      5                                                       Glx***      4.11      4                                                       Gly         3.30      3                                                       Ala         3.19      3                                                       Cys****     1.86      2                                                       Val         2.93      3                                                       Ile         1.00      1                                                       Leu         5.52      5                                                       Tyr         5.63      5                                                       Phe         2.29      2                                                       His         1.02      1                                                       Arg         4.54      4                                                       Pro         6.96      7                                                       Trp*        0.93      1                                                       ______________________________________                                         *hydrolyzed with 2.5 N NaOH, 110° C., 22 hours                         **Asn and Asp                                                                 ***Gln and Glu hydrolyzed with 2.5 N NaOH, 110° C., 22 hours           ****quantitated as Scarboxymethylcysteine                                

MEASURING CONDITIONS (1)

Column: YMC-R (φ4.6×250 mm)

Flow rate: 1 ml/min.

Eluent: A solution (water:acetonitrile:10% TFA=100 : 0:1), B solution(water:acetonitrile:10% TFA=40 : 60 : 1).

Concentration gradient: A/B=80/20 (0 min)→45/55 (70 min)→0/100 (70min)→0/100 (73 min).

Measurement wavelength: 220 nm.

On the other hand, the purified Gla¹⁷ human osteocalcin was subjected topurity assay by reverse phase HPLC under the measuring conditions (2)shown below.

MEASURING CONDITIONS (2)

Column: YMC-R (φ4.6×250 mm)

Flow rate: 1 ml/min.

Eluent: A solution (water:acetonitrile:10% TFA=100 : 0:1), B solution(water:acetonitrile:10% TFA =40 : 60 : 1).

Concentration gradient: A/B=100/0 (0 min)→100/0 (5 min)→0/100 (35min)→0/100 (40 min).

Measurement wavelength: 220 nm.

As the result, a single strong absorption was recognized based on thepeptide at a retention time of 30.7 min., and this was Gla¹⁷ humanosteocalcin of the present invention.

EXAMPLE 3 Synthesis of Glu¹⁷ human osteocalcin represented by theformula ##STR8##

The solid phase synthesis was carried out under the same conditions asin Example 2 except for using Boc-Glu(OBzl) in place of usingBoc-Gla(OcHex)₂ in introduction of the amino acid at the 17-position toobtain a protected peptide resin. A part (500 mg) of the protected resinwas HF decomposed in the same manner as in Example 2. The reduced typecrude Glu¹⁷ human osteocalcin obtained was oxidized, purified in thesame manner as in Example 2 to give 3.2 mg of Glu¹⁷ human osteocalcin.

According to the method of the present invention, introduction of Glacan be introduced more easily in Gla¹⁷ human osteocalcin and Glu¹⁷ humanosteocalcin, whereby chemical synthesis is rendered possible.

REFERENCE EXAMPLE PRODUCTION OF HUMAN BGP MEASURING TEST KIT USING HUMANBGP (a) PRODUCTION OF ANTI-BOVINE BGP MONOCLONAL ANTIBODY

After preparing a bound product of a bovine BGP extracted from bone ofbovine and purified to a purity of 95% or higher and a swinethyroglobulin, an emulsion was prepared with a Freund's completeadjuvant and immunized to mouse (Balb/c species). After confirmingappearance of antibody to bovine BGP in blood of mouse, cell suspensionwas prepared by using delivered spleen. Then, 1 to 5×10⁸ spleen cellswere fused with 2 to 10×10⁷ mouse myeloma cells (P3-X63-Ag8-U1) underthe conditions described in a literature (Galfre C, Milstein C., MethodsEnzymol. 1981; 73: 3-46). Hybridomas were cultivated in a Dulbecco'smodified-Eagle's medium containing 10% of bovine fetal serum andgeneration of anti-bovine BGP antibody was examined. Antibody forminghybridomas were cultivated to examine generation of anti-bovine BGPantibody. Cloning of antibody forming hybridoma was repeated twice ormore to obtain clones. By the method of inoculating cultivatedhybridomas to abdominal cavity of mouse (Balb/c species) and obtainingascites after 2 weeks, a large amount of monoclonal antibody wasobtained.

2. PRODUCTION OF ANTI-HUMAN BGP MONOCLONAL ANTIBODY

After preparing a bound product of Gla¹⁷ human osteocalcin with a purityof 95% or more obtained by chemically synthesizing and purifyingaccording to the method of Example 2 and a swine thyroglobulin,hybridoma was obtained in the same producing method of anti-bovine BGPmonoclonal antibody according to the above 1, and a large amount ofanti-human BGP monoclonal antibody was obtained according to the ascitesmethod.

3. PREPARATION OF POLYSTYRENE BEADS COVERED BY ANTI-HUMAN BGP MONOCLONALANTIBODY

Beads made of polystyrene (diameter: 6.3 mm) were dipped in a mouseascites purified IgG fraction solution of anti-human BGP monoclonalantibody prepared with a concentration of 16 μg/ml by using 0.3 ml of0.1 mole bicarbonate buffer (pH 9.6) per one bead, and allowed to standat room temperature overnight. Thereafter, the beads were washed with aphosphate buffer containing 0.1% Tween 20 (trade name) and then dried invacuum.

4. PRODUCTION OF ANTI-BOVINE BGP MONOCLONAL ANTIBODY LABELLED WITH ¹²⁵ I(IODINE)

According to a literature [Greenwood FC, Hunter WM, Glover JS., Biochem.J, 1963; 89: 114-123], IgG fraction of the anti-bovine BGP monoclonalantibody obtained from mouse ascites was labelled with ¹²⁵ I by theChloramine T method.

5. DETERMINATION OF HUMAN BGP

According to the so-called sandwich type radioactive immunoassay methodwherein the measurement was carried out by covering a solid substratewith a monoclonal antibody A and labelling a monoclonal antibody B with¹²⁵ I, determination of the human BGP was practiced. That is, in aplastic test tube, a 25 μl solution containing various concentration ofhuman BGPs was placed and then 200 μl of ¹²⁵ I labelled anti-bovine BGPmonoclonal antibody was placed, and further one polystyrene bead coveredby the anti-human BGP monoclonal antibody was placed therein, it wasshaked at room temperature for 3 hours, respectively. Next, after thereaction mixture was subjected to suction removal, the bead was washedwith 2 ml of purified water three times. A dose of radioactivity of ¹²⁵I labelled anti-bovine BGP antibody bound to the bead was measured byWell type gamma counter. The results are shown in Table 3. Finally, adose of radioactivity (cpm) against logarithm of a human BGPconcentration of a solution placed in a test tube was plotted againstlogarithm to prepare a dose-dependent curve. The resulting curve isshown in FIG. 4. If an unknown sample is measured in the same manner, aconcentration thereof can be known from the dose-dependent curve. BGPconcentrations of 66 normal sera were measured, and results ranging from3.1 ng/ml (Mean - 2SD) to 12.7 ng/ml (Mean+2SD) with an average of 6.2ng/ml were obtained.

                                      TABLE 3                                     __________________________________________________________________________                                       BGP con-                                   Test             Count (cpm)                                                                            B-Bo (cpm)                                                                             centration                                 tube No.                                                                           Content of test tube                                                                      Found                                                                             Average                                                                            Found                                                                             Average                                                                            (ng/ml)                                    __________________________________________________________________________     1   Total count 179630                                                                            179262                                                    2   Total count 178894                                                        3   Standard BGP 0 ng/ml                                                                      573 562                                                       4   Standard BGP 0 ng/ml                                                                      551                                                           5   Standard BGP 1 ng/ml                                                                      1495                                                                              1559 993 997                                              6   Standard BGP 1 ng/ml                                                                      1662     1060                                                 7   Standard BGP 4 ng/ml                                                                      4689                                                                              4830 4127                                                                              4268                                             8   Standard BGP 4 ng/ml                                                                      4970     4408                                                 9   Standard BGP 16 ng/ml                                                                     19487                                                                             19660                                                                              18925                                                                             19098                                           10   Standard BGP 16 ng/ml                                                                     19833    19271                                               11   Standard BGP 64 ng/ml                                                                     83474                                                                             83268                                                                              82912                                                                             82706                                           12   Standard BGP 64 ng/ml                                                                     83061    82499                                               13   Unknown specimen A                                                                        2969                                                                              3115 2407                                                                              2593 2.5                                        14   Unknown specimen A                                                                        3340     2778                                                15   Unknown specimen B                                                                        7062                                                                              7112 6500                                                                              6550 5.9                                        16   Unknown specimen B                                                                        7162     6600                                                17   Unknown specimen C                                                                        16261                                                                             16364                                                                              15699                                                                             15802                                                                              13.4                                       18   Unknown specimen C                                                                        16466    15904                                               __________________________________________________________________________     The meanings of abbreviations in Table 3 are as follows:                      Total count: count (cpm) of .sup.125 Ilabelled antiBGP monoclonal antibod     added into each assay tube.                                                   BBo: count (cpm) of antiBGP monoclonal antibody B complex bound by            insolubilized monoclonal antibody A.                                     

We claim:
 1. A method for preparing Gla¹⁷ human osteocalcin and Glu¹⁷human osteocalcin or salts thereof by the solid-phase peptide syntheticmethod, which comprises introducing γ-carboxyglutamic acid into theamino acid sequence of said osteocalcin by employing a protectedL-γ-carboxyglutamic acid represented by the formula: ##STR9## wherein nrepresents 0, 1 or 2, or a salt thereof in the appropriate position inthe reacting sequence of amino acids which form the osteocalcin.
 2. Theprocess for preparing human osteocalcin according to claim 1, whereinsaid Gla¹⁷ human osteocalcin and Glu¹⁷ human osteocalcin have thefollowing chemical formulae: ##STR10##
 3. The process for preparinghuman osteocalcin according to claim 1, wherein said salt of Gla¹⁷ humanosteocalcin or Glu¹⁷ human osteocalcin is a salt of an alkali metal, analkaline earth metal or an organic base.
 4. The process for preparinghuman osteocalcin according to claim 3, wherein said salt is selectedfrom the group consisting of sodium salt, potassium salt, lithium salt,calcium salt, magnesium salt, a salt of methylamine, ethylamine,diethylamine, triethylamine, benzylamine, pyrrolidine, piperidine,cyclohexylamine, dicyclohexylamine, ethanolamine, diethanolamine,ornithine, lysine and arginine.
 5. The process for preparing humanosteocalcin according to claim 1, wherein said solid phase methodcomprises binding valine at the C-terminal amino acid of a desired humanosteocalcin to an insoluble resin as a protected amino acid; binding aprotected amino acid successively from the C-terminal to form aprotected peptide chain; treating said protected peptide chain with areagent which eliminates side chain protective groups of the respectiveamino acids; eliminating side chain protective groups of the respectiveamino acids to give a peptide with the mercapto group of Cys beingdeprotected; oxidizing the peptide in a buffer to form an intramoleculardisulfide bond to obtain a crude human osteocalcin; and then purifyingthe crude human osteocalcin.
 6. The process for preparing humanosteocalcin according to claim 5, wherein said insoluble resin isselected from the group consisting of a chloromethyl resin, an oxymethylresin and a 4-(oxymethyl)phenylacetamidomethyl resin.
 7. The process forpreparing human osteocalcin according to claim 6, wherein said protectedpeptide has the following formula: ##STR11##
 8. The process forpreparing human osteocalcin according to claim 5, wherein the reactionof oxidizing the peptide in buffer is carried out by dissolving a crudehuman osteocalcin with mercapto group of Cys being deprotected in abuffer at an osteocalcin concentration of 10⁻³ to 10⁻⁷ mole/liter,adjusting pH to 6.0 to 8.5 and then stirring the solution at 4° to 50°C. for 4 to 30 hours.
 9. The process for preparing human osteocalcinaccording to claim 2, wherein purification is carried out by extraction,recrystallization, gel filtration chromatography, ion exchangechromatography, partition chromatography, adsorption chromatography,reverse phase chromatography, electrophoresis or countercurrentpartition.